Characterization of the Serine Protease TlSP1 from Trichoderma longibrachiatum T6 and Its Function in the Control of Heterodera avenae in Wheat

Serine protease is an extracellular protease secreted by biocontrol fungi that can effectively control nematode diseases by degrading nematode eggshells and enhancing plant resistance. <i>Trichoderma longibrachiatum</i> T6, an important biocontrol fungus, has been demonstrated to effectively parasitize and degrade <i>Heterodera avenae</i> cysts, eggs, and second-stage juveniles (J2s). However, the genes that encoding serine protease and their functions in <i>T. longibrachiatum</i> T6 have not been thoroughly investigated. In this study, we successfully cloned and sequenced the serine protease gene <i>TlSP1</i> in <i>T. longibrachiatum</i> T6. Our results revealed that the expression level of the <i>TlSP1</i> gene was induced and significantly increased in <i>T. longibrachiatum</i> T6 after inoculation with <i>H. avenae</i> cysts. The full-length sequence of the coding region (CDS) of <i>TlSP1</i> gene was 1230 bp and encoded a protein consisting of 409 amino acids. Upon the transformation of the <i>TlSP1</i> gene into <i>Pichia pastoris</i> X33, the purified recombinant TlSP1 protein exhibited optimal activity at a temperature of 50 °C and pH 8.0. Following 4-10-day of treatment with the purified recombinant TlSP1 protein, the eggshells and content were dissolved and exuded. The number of nematodes invading wheat roots was reduced by 38.43% in the group treated with both TlSP1 and eggs on one side (P1+N) compared to the control group, while the number of nematodes invading wheat roots was reduced by 30.4% in the TlSP1 and eggs two-sided treatment group (P1/N). Furthermore, both the P1+N and P1/N treatments significantly upregulated genes associated with defense enzymes (<i>TaPAL</i>, <i>TaCAT</i>, <i>TaSOD</i>, and <i>TaPOD</i>), genes involved in the lignin synthesis pathway (<i>TaC4H</i>, <i>Ta4CL2</i>, <i>TaCAD1</i>, and <i>TaCAD12</i>), and salicylic acid (SA)-responsive genes (<i>TaNPR1</i>, <i>TaPR1</i>, and <i>TaPR2</i>) and led to the high expression of jasmonic acid (JA)-responsive genes (<i>TaPR4</i>, <i>TaOPR3</i>, and <i>TaAOS2</i>). This study has highlighted the significant role of the <i>TlSP1</i> gene in facilitating <i>H. avenae</i> eggshells' dissolution, preventing nematode invasion in the host plant, and boosting plant resistance in wheat.